Human Adipose Tissue Lysate-Based Hydrogel for Lasting Immunomodulation to Effectively Improve Spinal Cord Injury Repair.

The long-term inflammatory microenvironment is one of the main obstacles to inhibit acute spinal cord injury (SCI) repair. The natural adipose tissue-derived extracellular matrix hydrogel shows effective anti-inflammatory regulation because of its unique protein components. However, the rapid degradation rate and removal of functional proteins during the decellularization process impair the lasting anti-inflammation function of the adipose tissue-derived hydrogel. To address this problem, adipose tissue lysate provides an effective way for SCI repair due to its abundance of anti-inflammatory and nerve regeneration-related proteins. Thereby, human adipose tissue lysate-based hydrogel (HATLH) with an appropriate degradation rate is developed, which aims to in situ long-term recruit and induce anti-inflammatory M2 macrophages through sustainedly released proteins. HATLH can recruit and polarize M2 macrophages while inhibiting pro-inflammatory M1 macrophages regardless of human or mouse-originated. The axonal growth of neuronal cells also can be effectively improved by HATLH and HATLH-induced M2 macrophages. In vivo experiments reveal that HATLH promotes endogenous M2 macrophages infiltration in large numbers (3.5 × 105/100 µL hydrogel) and maintains a long duration for over a month. In a mouse SCI model, HATLH significantly inhibits local inflammatory response, improves neuron and oligodendrocyte differentiation, enhances axonal growth and remyelination, as well as accelerates neurological function restoration.

Synthesis and evaluation of imidazo[1,2-a]pyrazine derivatives as small molecule Gαq/11 inhibitors against uveal melanoma.

Uveal melanoma (UM) is an aggressive malignancy with high mortality in adults and lacks effective systemic therapies. Activating gene mutations related to the Gαq/11 signaling pathway are prevalent in UM, and Gαq/11 inhibitors have shown anti-UM activity in vitro and in vivo. In this study, we designed and synthesized a series of imidazo[1,2-a]pyrazine derivatives as Gαq/11 inhibitors, and discovered GQ352 with the selective antiproliferative activity against UM cells. Importantly, GQ352 directly binds to the Gαq and inhibits the dissociation of Gαßγ heterotrimers with the IC50 value of 8.9 µM. GQ352 inhibits UM tumorigenesis by suppressing Gαq/11 downstream ERK phosphorylation and YAP dephosphorylation, as shown in Western blot analysis. In addition, GQ352 displayed reasonable physiochemical properties and human liver microsome stability, indicating the potential application in UM treatment.

Discovery of small molecule Gαq/11 protein inhibitors against uveal melanoma.

Constitutively activated G proteins caused by specific mutations mediate the development of multiple malignancies. The mutated Gαq/11 are perceived as oncogenic drivers in the vast majority of uveal melanoma (UM) cases, making directly targeting Gαq/11 to be a promising strategy for combating UM. Herein, we report the optimization of imidazopiperazine derivatives as Gαq/11 inhibitors, and identified GQ262 with improved Gαq/11 inhibitory activity and drug-like properties. GQ262 efficiently blocked UM cell proliferation and migration in vitro. Analysis of the apoptosis-related proteins, extracellular signal-regulated kinase (ERK), and yes-associated protein (YAP) demonstrated that GQ262 distinctly induced UM cells apoptosis and disrupted the downstream effectors by targeting Gαq/11 directly. Significantly, GQ262 showed outstanding antitumor efficacy in vivo with good safety at the testing dose. Collectively, our findings along with the favorable pharmacokinetics of GQ262 revealed that directly targeting Gαq/11 may be an efficient strategy against uveal melanoma.

Design, Synthesis, and Evaluation of Small Molecule Gαq/11 Protein Inhibitors for the Treatment of Uveal Melanoma.

Uveal melanoma is the ocular malignancy and mainly driven by oncogenic mutations of Gαq/11 proteins. Previous targeted therapy for melanoma treatment was limited to specific downstream signaling pathway, and inhibiting the "molecular switches" G proteins for melanoma treatment therapy was rarely described. We herein report the discovery of imidazopiperazine derivatives as Gαq/11 protein inhibitors. The most promising compound GQ127 showed good efficacy and safety in inositol monophosphate (IP1) assay by directly inhibiting Gαq/11 proteins. GQ127 induced uveal melanoma cells apoptosis and displayed potent antitumor activities in uveal melanoma cells viability, migration, and invasion. The effects of GQ127 on Gαq/11 signaling pathway were confirmed by analyzing the downstream effectors yes-associated protein (YAP) and extracellular signal-regulated kinase (ERK). More importantly, GQ127 significantly suppressed UM xenograft growth in mouse model without severe toxicity at the testing dose. These findings provide a lead compound that directly targets the Gαq/11 proteins for uveal melanoma treatment.

Cleavable and tunable cysteine-specific arylation modification with aryl thioethers.

Cysteine represents an attractive target for peptide/protein modification due to the intrinsic high nucleophilicity of the thiol group and low natural abundance. Herein, a cleavable and tunable covalent modification approach for cysteine containing peptides/proteins with our newly designed aryl thioethers via a S N Ar approach was developed. Highly efficient and selective bioconjugation reactions can be carried out under mild and biocompatible conditions. A series of aryl groups bearing different bioconjugation handles, affinity or fluorescent tags are well tolerated. By adjusting the skeleton and steric hindrance of aryl thioethers slightly, the modified products showed a tunable profile for the regeneration of the native peptides.

AUXIN RESPONSE FACTOR 1 Acts as a Positive Regulator in the Response of Poplar to Trichoderma asperellum Inoculation in Overexpressing Plants.

Numerous Trichoderma strains have been reported to be optimal biofertilizers and biocontrol agents with low production costs and environmentally friendly properties. Trichoderma spp. promote the growth and immunity of plants by multiple means. Interfering with the hormonal homeostasis in plants is the most critical strategy. However, the mechanisms underlying plants' responses to Trichoderma remain to be further elucidated. Auxin is the most important phytohormone that regulates almost every aspect of a plant's life, especially the trade-off between growth and defense. The AUXIN RESPONSE FACTOR (ARF) family proteins are key players in auxin signaling. We studied the responses and functions of the PdPapARF1 gene in a hybrid poplar during its interaction with beneficial T. asperellum strains using transformed poplar plants with PdPapARF1 overexpression (on transcription level in this study). We report that PdPapARF1 is a positive regulator for promoting poplar growth and defense responses, as does T. asperellum inoculation. PdPapARF1 also turned out to be a positive stimulator of adventitious root formation. Particularly, the overexpression of PdPapARF1 induced a 32.3% increase in the height of 40-day-old poplar plants and a 258% increase in the amount of adventitious root of 3-week-old subcultured plant clones. Overexpressed PdPapARF1 exerted its beneficial functions through modulating the hormone levels of indole acetic acid (IAA), jasmonic acid (JA), and salicylic acid (SA) in plants and activating their signaling pathways, creating similar results as inoculated with T. asperellum. Particularly, in the overexpressing poplar plants, the IAA level increased by approximately twice of the wild-type plants; and the signaling pathways of IAA, JA, and SA were drastically activated than the wild-type plants under pathogen attacks. Our report presents the potential of ARFs as the crucial and positive responders in plants to Trichoderma inducing.

Macular choroidal thickness in Chinese preschool children: decrease with axial length but no evident change with age.

AIM: To explore the distribution pattern of macular choroidal thickness (ChT) and its association with age as well as refractive status in Chinese preschoolers. METHODS: School-based, cross-sectional study. A total of 550 healthy preschool children aged 3 to 6 years old from 6 kindergartens were enrolled. Comprehensive ocular examinations, including measurement of visual acuity, axial length, intraocular pressure and slit-lamp examination before cycloplegia, as well as refraction measurement and swept-source optical coherence tomography (SS-OCT) examination after cycloplegia, were performed. The macular ChT in each sector of the ETDRS grid was measured by the built-in software of SS-OCT. RESULTS: The mean central ChT of the participants was 312±59 µm. The mean axial length and spherical equivalent refraction were 22.36±0.72 mm and 1.51±0.83 D, respectively. Axial length increased with age (P<0.001), while the spherical equivalent refraction was similar among different age groups. Similarly, no significant difference was observed in ChT in all sectors among different age groups (all P>0.05). The central ChT of 3-4, 5 and 6 years old children was 314±59 µm, 312±60 µm and 312±59 µm, respectively (P=0.920). No difference was observed in ChT in most of the sectors between genders. No statistical significant difference was observed among different refractive groups (all P>0.05), though the ChT of each sector seemed to be smaller in myopic children. Axial length and weight were the independent factors of central ChT. Children with longer axial length (ß=-21.184, P<0.001) and smaller weight (ß=1.502, P=0.041) tended to have thinner choroid. CONCLUSION: In preschool children, the ChT remains relatively stable with age, while a negative association between ChT and axial length existed. This will be helpful to elucidate the characteristics of ChT during the early refractive development.

Lindera cyclopentenedione intermediates from the roots of Lindera aggregata.

Chromatographic fractionation of the roots of Lindera aggregata has led to the isolation of three new monomers of Lindera cyclopentenedione derivatives (1-3), a pair of new enantiomers of bi-linderone derivatives (4a/4b), and six known Lindera cyclopentenediones (5-8 and 9a/9b). Their structures were determined by NMR and MS data. The absolute configurations of the new bi-linderone derivative enantiomers (4a/4b) were determined by ECD calculation. (±)-Lindepentone A (1) presents the novel skeleton of 3,5-dioxocyclopent-1-enecarboxylate. Lindoxepines A (2) and B (3) present an unprecedented oxepine-2,5-dione derivative skeleton, which may be enlightening for the in vivo biosynthesis of the monomers of Lindera cyclopentenediones. A possible biosynthetic pathway for 1 and a plausible biosynthetic pathway from stilbenes to Lindera cyclopentenediones via the key intermediates 2 and 3 were postulated. The inhibitory activity of these compounds against three microorganisms was also evaluated.

A quantitative comparison of five optical coherence tomography angiography systems in clinical performance.

AIM: To compare the clinical performance of 4 spectral-domain (SD) optical coherence tomography angiography (OCTA) systems: AngioVue™, AngioPlex™, Spectralis® OCTA, AngioScan, and 1 swept-source (SS) OCTA SS OCT Angio™. METHODS: Twenty-seven undilated right eyes of 27 participants underwent OCTA examination using five different systems respectively for both 3×3 and 6×6 mm2 scan pattern (Spectralis OCTA for 3×3 mm2 scan only). Image quality, including vessel valid visibility and the number of motion artifacts, and acquisition time were evaluated. Repeated measures analysis of variance (ANOVA) with Bonferroni's post-test and Friedman test with Dunn's post-test were used to compare measurements. RESULTS: The age of the subjects was 28.19±5.55y (range, 23-49y). The spherical equivalent refraction was -2.55±1.84 D (range, 0.00 to -5.25 D). Significant difference was observed in the evaluation of vessel valid visibility (AngioVue the highest: 0.111±0.031 for 3×3 mm2 scan and 0.128±0.020 for 6×6 mm2 scan), number of motion artifacts (AngioVue the fewest: 0.778±1.086 for 3×3 mm2 scan and 0.333±0.620 for 6×6 mm2 scan) and acquisition time (AngioPlex the shortest: 8.537±1.921s for 3×3 mm2 scan and 8.298±1.741s for 6×6 mm2 scan; all P<0.001). CONCLUSION: There is poor agreement of measurements among systems. AngioVue provides images with the highest vessel valid visibility and the fewest motion artifacts. AngioPlex achieves the shortest acquisition.

Efficiency and Safety of ß-CD-(D3)7 as siRNA Carrier for Decreasing Matrix Metalloproteinase-9 Expression and Improving Wound Healing in Diabetic Rats.

Overexpression of matrix metalloproteinase-9 (MMP-9) is critical for diabetic chronic wounds involved in the refractory wound healing process. We aimed to develop a strategy through RNAi to decrease MMP-9 expression and improve diabetic wound healing. We had explored ß-CD-(D3)7 as a gene carrier to take siRNA and effectively interfere with MMP-9 expression. It has been proven that ß-CD-(D3)7 could be used as an effective siRNA delivery system. In this study, we want to know about the efficiency and safety of ß-CD-(D3)7/MMP-9 siRNA for improving wound healing in diabetic rats. ß-CD-(D3)7/MMP-9 siRNA treated animals show lower levels of MMP-9 expression, which induce faster wound-close rates. Histological evaluation indicates that ß-CD-(D3)7/MMP-9 siRNA significantly increases the content of collagen around the injured tissues. The number of neutrophilic ganulocytes was significantly decreased through treatment of ß-CD-(D3)7/MMP-9 siRNA. In vivo fluorescence imaging assessment shows that ß-CD-(D3)7/MMP-9 siRNA could not cause organ damage and organ accumulation. The results suggest that ß-CD-(D3)7/MMP-9 siRNA might be developed as a novel topical agent for the diabetic wounds treatment.

ATP triggered drug release and DNA co-delivery systems based on ATP responsive aptamers and polyethylenimine complexes.

Stimuli-responsive nanocarriers for anticancer drug and gene co-delivery are a promising strategy in cancer therapy due to their combination of chemotherapy and gene therapy. In this work, we developed a facile and effective method to fabricate stimuli-responsive nanocarriers for anticancer drug and gene co-delivery based on complexes of polyethylenimine (PEI) with an adenosine triphosphate (ATP) responsive aptamer duplex (ARAD). No chemical reactions or complex modifications were used in the construction processes. In this system, Doxorubicin-loaded aptamer duplex and plasmid DNA (p53) can be bound by PEI by electronic interactions to form stable complexes which effectively protect the aptamer and p53 from DNase degradation. The intercalated Dox can be released on-demand by a structural change in the aptamer duplex in an ATP-rich environment. The morphology and average size of the nanocarriers were characterized by zeta potential and transmission electron microscopy (TEM). The nanocarriers exhibit lower cell toxicity in HeLa cell lines relative to PEI. RT-PCR and Western blot analysis confirmed that p53 could be effectively delivered and expressed in HeLa cells by PEI/ARAD/p53 complexes. Moreover, the apoptosis percentage of HeLa cells treated with PEI/ARAD/Dox/p53 complex increased to 40.8%, compared to 24.7% for PEI/ARAD/Dox complex and 11.5% for PEI/ARAD/p53, respectively. The result demonstrated that the combinatorial delivery of Dox and p53 by nanocarriers could induce synergistic actions and lead to effective cancer cell apoptosis.

Cationic star-shaped polymer as an siRNA carrier for reducing MMP-9 expression in skin fibroblast cells and promoting wound healing in diabetic rats.

BACKGROUND: Excessive expression of matrix metalloproteinase-9 (MMP-9) is deleterious to the cutaneous wound-healing process in the context of diabetes. The aim of the present study was to explore whether a cationic star-shaped polymer consisting of ß-cyclodextrin (ß-CD) core and poly(amidoamine) dendron arms (ß-CD-[D3]7) could be used as the gene carrier of small interfering RNA (siRNA) to reduce MMP-9 expression for enhanced diabetic wound healing. METHODS: The cytotoxicity of ß-CD-(D3)7 was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MMT) method in the rat CRL1213 skin fibroblast cell line. The transfection efficiency of ß-CD-(D3)7/MMP-9-small interfering RNA (siRNA) complexes was determined by confocal microscopy and flow cytometry. Quantitative real time (RT) polymerase chain reaction was performed to measure the gene expression of MMP-9 after the transfection by ß-CD-(D3)7/MMP-9-siRNA complexes. The ß-CD-(D3)7/MMP-9-siRNA complexes were injected on the wounds of streptozocin-induced diabetic rats. Wound closure was measured on days 4 and 7 post-wounding. RESULTS: ß-CD-(D3)7 exhibited low cytotoxicity in fibroblast cells, and easily formed the complexes with MMP-9-siRNA. The ß-CD-(D3)7/MMP-9-siRNA complexes were readily taken up by fibroblast cells, resulting in the downregulation of MMP-9 gene expression (P<0.01). Animal experiments revealed that the treatment by ß-CD-(D3)7/MMP-9-siRNA complexes enhanced wound closure in diabetic rats on day 7 post-wounding (P<0.05). CONCLUSION: ß-CD-(D3)7 may be used as an efficient carrier for the delivery of MMP-9-siRNA to reduce MMP-9 expression in skin fibroblast cells and promote wound healing in diabetic rats.

Efficient gene transfection in the neurotypic cells by star-shaped polymer consisting of ß-cyclodextrin core and poly(amidoamine) dendron arms.

In order to develop the effective vectors that had high gene transfection capability and low cytotoxicity in the neuronal cells, we tested the star-shaped polymer consisting of ß-cyclodextrin core and poly(amidoamine) (PAMAM) dendron arms [ß-CD-(D3)7] as the vector to transfect the human neuroblastoma SH-SY5Y cells. The physicochemical properties of the ß-CD-(D3)7/plasmid DNA (pDNA) complexes were characterized by using gel electrophoresis, dynamic light scattering, transmission electron microscopy and zeta-potential experiments. Among the human neuroblastoma SH-SY5Y cells, ß-CD-(D3)7/pDNA complex demonstrated a lower toxicity compared to those of PAMAM (G=4, with an ethylenediamine core)/pDNA complex. When the N/P ratio was over 20, it was observed that PAMAM had a faster increment in toxicity compared to ß-CD-(D3)7. Fluorescent image, confocal microscopy image and flow cytometry showed that ß-CD-(D3)7/pDNA complexes had significantly higher transgene activity than that of PAMAM/pDNA complexes. For example, the transfection efficiency was 20% and 7.5% for ß-CD-(D3)7/pDNA and PAMAM/pDNA complexes, respectively. These results indicated that ß-CD-(D3)7 might be a promising candidate for neurotypic cells gene delivery with the characteristics of good biocompatibility, relatively high gene transfection capability and potential in vivo gene delivery ability.